In this Phase I SBIR project, Parabon NanoLabs, Inc. (PNL) will produce a novel, nano- pharmaceutical compound that preferentially selects and destroys brain tumor stem cells (BTSCs) to aid the treatment of glioblastoma multiforme (GBM), one of the most lethal brain cancers. BTSCs in GBM have been identified as a highly tumorigenic cell subpopulation that promotes tumor angiogenesis and therapeutic resistance. For example, as few as 100 BTSCs can initiate tumor growth in a mouse model, whereas 1 million non-stem cells from the same tumor cannot. The inability to eliminate sufficient BTSCs with the current standard of care may account for the >90% recurrence rate of GBM and its poor prognosis. Using Parabon's Essemblix Drug Development Platform, the lead compound and its experimental variants will be developed upon a proprietary molecular breadboard, called PNL24, that can be functionalized with different targeting ligands and cytotoxic payloads in plug and play fashion. These compounds will be used to test the hypothesis that mixed-ligand, low-affinity targeting can achieve superior BTSC targeting specificity versus single-ligand alternatives. To test BTSC specificity, three targeting compounds will be created by functionalizing PNL24 with one of two different targeting ligands and a combination of both. Dye-labeling of each compound will be used to test in vitro targeting specificity, measured via fluorescent confocal microscopy, against BTSC and normal human cell lines serving as controls, specifically, neural stem cells, neurons and astrocytes. The most selective of the three targeting structures (hypothetically the mix-ligand construct) wil be further functionalized with a diphtheria toxin derivative and the result tested for efficacy against BTSC and safety against three normal cell lines via standard cytotoxicity assay. Finally, the anti-BTSC efficacy of this compound will be tested in a hBTSC intracranial xenograft mouse model via convection-enhanced delivery (CED). If found to be both safe and effective, this compound will be the lead compound for future studies in a subsequent Phase II project that examines systematic toxicity, PK and biodistribution in preparation for an eventual IND application.